GRIFFIN,
COHEN, A N D SCHECHTER
The Assignment of an Exchangeable Low-Field N H Proton Resonance of Ribonuclease A to the Active-Site Histidine-1 19t John H. Griffin,* Jack S. Cohen, and Alan N. Schechter
High-resolution proton magnetic resonance spectra between 11 and 14 ppm of bovine ribonuclease A in H 2 0show that a single resonance titrates with changes in pH. This resonance arises from a histidine ring nitrogen proton and exhibits an apparent pK, of 5.8. This pKa value can be correlated with that of only one of the four histidine ring C-2 protons, namely, that which has been assigned to the active site histidine-119. Additional evidence for the correlation is obtained from the ABSTRACT:
H
igh-resolution proton magnetic resonance (pmr) spectroscopy can provide information about the environment of individual atoms within protein molecules if the resonances can be resolved and assigned to specific nuclei. For pmr studies of proteins, DzO is routinely used as the solvent in order to avoid a dominant HzO proton resonance and to simplify the protein pmr spectrum by exchanging deuterons for protons bound to oxygen and nitrogen atoms. Nonetheless, recent reports show that individual resonances of exchangeable protons can be resolved at low field between 10 and 15 ppm in the pmr spectra of lysozyme (Glickson et af., 1971), myoglobin (Patel et al., 1970a; Sheard et al., 1970), hemoglobin (Patel et al., 1970b), and RNase A’ (Patel et al., 1972) in HzO solutions. Several of these low-field resonances in lysozyme have been assigned to specific indole N H protons of tryptophan residues (Glickson et af., 1971). Based on the proposed assignments of the four histidine ring C-2 proton resonances to specific histidine residues (Meadows et al., 1968), we report here the assignment of an imidazole NH proton resonance of bovine pancreatic RNase A to the activesite histidine-119 on the basis of the determination of its apparent pK, in the free enzyme at different temperatures and in the enzyme complexed with each of four different inhibitors. Materials and Methods Bovine pancreatic RNase A was obtained from Worthington Biochemical Corp. (Rutherford, N. J.) as a phosphatefree powder. Concentrated solutions of RNase A were dialyzed extensively against deionized water and then lyophilized. Solutions for pmr studies were then made by dissolving the RNase A to 100 mg/ml in HzO containing 0.1 M NaCI. Concentrated HCl and NaOH were used for pH adjustments. 2‘-CMP, 3’-CMP, and 5’-CMP were obtained from Sigma Chemical Co. (St. Louis, Mo.). Pmr spectra were recorded on a Varian HR 220 nmr spec-
7 From the Laboratory of Chemical Biology, National Institute of Arthritis, Metabolic and Digestive Diseases, and the Physical Sciences Laboratory, Division of Computer Research and Technology, National Institutes of Health, Bethesda, Maryland 20014. Received Nouember 30, 1972. Supported by a Helen Hay Whitney Foundation Fellowship to J. H. G . I Abbreviation used is: RNase A, bovine pancreatic ribonuclease A .
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selective shifts of the apparent pK, values caused by temperature variation and by the binding of the inhibitors cytidine 2‘monophosphate, cytidine 3’-monophosphate, cytidine 5’monophosphate, and inorganic phosphate. These results suggest that a specific histidine ring nitrogen proton of residue 119 is relatively inaccessible to solvent. Solvent inaccessibility of histidine residues as defined by proton magnetic resonance studies of proteins in HzO is discussed.
trometer. A Varian C1024 time-averaging computer was used to accumulate spectra for 1-5 hr at each pH value. The probe temperature was 22 O unless otherwise noted. The chemical shift values are given relative to external tetramethylsilane in carbon tetrachloride solution. pH values were measured in the pmr sample tube using a long thin combination electrode and a Radiometer Model 26 pH meter. The pH was determined before and after each spectrum was recorded, with these values differing by less than 0.04 pH unit. The final pH value is quoted in this paper. The apparent pKa values exhibited by the imidazole NH resonance were generally determined as the pH at half-maximal chemical shift change. However, the titration data obtained in the presence of 0.1 M Pi were computer curve fitted to yield apparent pK, values of the several titrating groups (Shrager et al., 1972). Results and Discussion Three pmr spectra of RNase A in H 2 0 at three different pH values are shown in Figure 1 . Pmr spectra of RNase A in DzO at pH 5 or 9 contain no resonances between 10 and 13 ppm, and, therefore, all resonances in Figure 1 belong to exchangeable protons. One resonance shifts from 12.9 ppm at pH 4.4 to 11.1 ppm at pH 8.9, and it follows a simple titration curve with an apparent pKa of 5.8. From the observed pKa, the chemical shift values and the total change in chemical shift due to pH changes in model compounds, this resonance has been assigned by Patel et al. (1972) to an imidazole NH proton which exchanges very slowly with solvent H 2 0 .We have found that the pH dependences (see below, Figure 3) of the other exchangeable resonances shown in Figure 1 are generally similar to the results of Patel er al. (1972). To which one of the four histidine residues of RNase A does the slowly exchanging NH proton belong? The pmr titration curves of the four imidazole C-2 proton resonances have been described, and an assignment of each resonance to a corresponding histidine residue in the RNase structure has been proposed (Meadows et a[., 1968). Therefore, direct comparison of the pKa value exhibited by the imidazole NH proton resonance to the pK, values exhibited by the imidazole C-2 proton resonances in H 2 0 should allow assignment of the NH proton resonance to a specific histidine residue. In Figure 2, that direct comparison is made by normalizing the pH-
ASSIGNMENT OF NH RESONANCE IN PMR OF
RNASEA I
I
I
I
I
I
I
C2-H (0,A,0 )
- 12.9
0
4
-
& v)
-I
a
P
w
I 0
FIGURE 1: Pmr spectra of RNase A in H20,q.l M NaCl, 22", at pH 4.4,5.3, and 8.9. Different numbers of scans were averaged to obtain each spectrum using the Varian C1024 computer.
dependent chemical shift values of the N H and the C-2 proton resonances. The titration of the NH proton resonance coincides exactly with the titration of the C-2 proton resonance, H-3, which has been assigned to histidine-119 in the active site of RNase (Meadows et al., 1968).2Assuming the proposed assignment for the C-2 proton resonances are correct, the slowly exchanging imidazole NH proton is assigned to histidine-119. A comparison of the pH dependence of the imidazole N H resonance with that of the C-2 proton resonances was conducted at 33" in order to compare the apparent pKa values under a different set of solution conditions. At 33", 0.1 M NaCl, the apparent pKa value of both the imidazole NH resonance and the C-2 proton resonance, H-3, was 5.5 while the C-2 proton resonances H-1 and H-2 exhibited apparent pK, values of 6.85 and 6.2, respectively. Thus, again the apparent pK, value of the imidazole NH resonance correlates only with that of the C-2 proton resonance of histidine-1 19. Additional evidence for assignment of the imidazole NH proton resonance to the histidine residue which exhibits the C-2 proton resonance, H-3, i.e., to histidine-1 19, comes from studying the binding of the inhibitors 2'-CMP, 3'-CMP, 5'CMP, and inorganic phosphate. Meadows et al. (1969) showed that the binding of these inhibitors causes selective shifts of the imidazole C-2 proton titration curves. As seen in' Figure 3, the different cytidine mononucleotide inhibitors cause selective shifts of the imidazole NH resonance titration curve. At pH 2.5, where inhibitor binding is negligibly small, the imidazole NH proton resonates at 12.9 ppm. As the binding of inhibitor increases when the p H increases from 2.5 to 4.5, the chemical shift of the imidazole NH proton in the protonated histidine moves downfield by 0.4 ppm for 3'-CMP and 5'-CMP and by more than 0.8 ppm for 2'-CMP. The
I).'
30
5.0
9.0
7.0
PH
FIGURE 2: Pmr data for titration of histidine residues in RNase A in HzO, 0.1 M NaC1,22". Chemical shift changes of the NH resonance
are normalized with the chemical shift changes of the C-2-H resonances to facilitate comparison. The designations of the C-2 proton resonances as H-1, H-2, and H-3 follow the convention of Meadows et al. (1 968).
apparent pK. values and the changes in chemical shift of the protonated histidine exhibited in Figure 3 are tabulated in Table I for comparison with the values of the imidazole C-2 proton resonances (Meadows et al., 1969). The different apparent pK, values of the imidazole NH resonance in the different RNase-inhibitor complexes correlate only with those of histidine-119. Inhibitor binding causes a downfield shift of the NH resonance. It should also be noted that inhibitor binding causes downfield shifts in the C-2 proton resonance of the protonated histidine-119, but upfield shifts in the C-2 proton resonance of the protonated histidine-12. Both the
TABLE I : Comparison of Apparent pKa Values of RNase Imidazole N H and C-2 Proton Resonances in Complexes with CMPInhibitors."
NH Resonance
H-3 Resonancec H-2 Resonancec His-119d His-12
Inhibitor PK,
A6 (ppm)
PKa
3'-CMP 7.2 2'-CMP > 8 . 0 5'-CMP 6.2
-0.4 -0.86 -0.4
7.4 >8.0
